Biocontrol Methods in Avoidance and Downsizing of Mycotoxin Contamination of Food Crops
, Ksenija Markov 2, Tina Lešić 3, Jadranka Frece 2, Danijela Petrović 4 and Jelka Pleadin 3,*Round 1
Reviewer 1 Report
Process - 1614701
Title: Bio-control methods in prevention and reduction of mycotoxin contamination of seeds and cereals.
General Impression:
This review paper summarizes existing biocontrol methods aimed at reduction and/or prevention of mycotoxins and their associated fungi found predominantly in/on cereal seeds. Included are the most relevant mycotoxins and their respective toxic effects in specific commodities. Pre-harvest biocontrol methods and post-harvest biocontrol methods are discussed. The authors hope the information may be helpful in increasing sustainability of agriculture pertaining to food and feed production. The information is current and relevant, addressing worldwide mycotoxin concerns and should be of interest to a wide audience.
The manuscript is relatively well written, needing minor stylistic edits and inclusion of few details for clarity. Please see the specific comments section.
Specific comments/suggestions:
Line 46: should read…are viewed as one of the most dangerous…
Line 47-48: should read…Mycotoxins are secondary metabolites of mostly fungal genera, including the Aspergillus, Fusarium, and Penicillium species.
Line 54: should read…fungal infestation…
Line 57: should read…the accumulation of toxic substances.
Line 64: should read…easily attack damaged kernels,
Line 68: should read…challenges in successful crop…
Line 69: should read…seedling blight…
Line 72: should read…disease management.
Line 81-82: should read…can play a key role in minimizing mycotoxin contamination by controlling mould growth and subsequent mycotoxin biosynthesis.
Line 92-96: should read…Moreover, the economic aspects of measures taken against moulds and mycotoxins are not negligible and cannot be overlooked. As a result of climatic changes, these measures must be adapted in order to remain efficient, but need to also be cost effective [21-23]. Given that biocontrol methods use natural sources and do not require additional care after being used, they are considered promising. (From what perspective are they considered promising? Economic perspective?? They may be costly to develop. This may need clarification.)
Line 97: should read…possibility of implementing biocontrol…
Line 99-100: should read…with mycotoxins, to the ultimate goal of 99 increasing the sustainability of the agricultural chain in food and feed production.
Line 103: should read…in an accumulative process. Their…
Line 116: should read…has been modified,
Line 120: should read…Pre-harvest biocontrol methods
Line 123-126: should read…Innovations introduced in the pre-harvest stage can be attributed to plant genetic engineering as a biotechnology advancement in mould-produced mycotoxin control. Plant genetic engineering aimed at the development of mycotoxin-resistant varieties and crop detoxification, places the emphasis on the fungi and mycotoxins most relevant from…
Line 128-129: should read…The most reasonable approach to the prevention of mycotoxin contamination is by breeding cereals and other crops resistant to moulds and therefore not prone to accumulating mycotoxins.
Line 136: should read…to infection by mycotoxin producing fungi. (Mycotoxins do not infect, they accumulate…fungi infect or colonize and produce mycotoxins. Did you mean ‘mycotoxin accumulation’? This is an important distinction. Need to clarify!)
Line 147-148: should read…a milder infection, and lower mycotoxin levels, in the harvested product…
Line 149: should read…in maize ears;
Line 151-152: should read…to create a transgenic maize line. Upon subsequent inoculation with Aspergillus flavus sufficiently concentrated to infect the transgenic maize ears, AF was not detected…(please use ‘ears’ instead of ‘cobs’)
Line 154: …gave high protection levels in field trials…(against what?? Fusarium? Fumonisin?? Please complete the sentence)
Line 156: …reduced disease severity (what disease? Please clarify)
Line 159-160: …this disease… (what disease? Please clarify)
Line 161-162: incomplete sentence…should perhaps read??... Karlovsky [55], Duvick [56], and Munkvold [57] reviewed a variety of genetic-based approaches that are being or have been used for mycotoxin control. (Please make sure the sentence reads as you intended, if not, please correct it.)
Line 162: should read… One of the approaches…
Line 163: reduce infection…(fungal infection? Please clarify.)
Line 179: should read… as compared to the parent maize.
Line 182: Additionally, reductions of aflatoxin production in Bt hybrids were not observed. (Are you sure? Do you have a reference for this statement??)
Line 186: should read… remains controversial.
Lines 205-206: should read… In the last few decades, the interest in PBM use for increasing yields and resilience of agricultural crops has been steadily growing…
Lines 213-214: should read… Increase in grain yield and stability was associated with seed coating formulation with the AM fungi and Trichoderma consortium with reduced or withheld fertilizer…
Line 223: should read…to maintain crop health and vigor.
Line 328: should read… Abbas…
Line 345: should read… maize was also reported [131]. Additionally, the inhibitory effect…
Line 368: should read… nontoxigenic F. verticillioides…
Line 369-370: should read… and inhibiting toxin synthesis.
Line 373-374: should read… which resulted in prevention of accumulation of mycotoxins during storage.
Line 384: should read… Post-harvest biocontrol methods
Line 388-390: should read… is that they do not significantly change the physicochemical properties of the cereals and leave any toxic mycotoxin residue behind [121,147]. In addition, the cost of …
Line 392: should read… more mycotoxin…
Line 396: should read… if that is not possible, one has to ensure to…
Line 418: should read…affected the conidia producing ability of the fungus.
Line 422: should read…inhibited the growth…
Line 423-424: should read…Evidence from microscopy and quantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that,…
Line 433: What are ‘cellular laches’?
Line 439: should read…phenolic compounds,…
Line 444: should read…type of acid, concentration, the…
Line 452: should read…reported antifungal…
Line 455: should read…needed to elucidate the mechanism…
Line 458-459: should read…Some higher plants produce secondary metabolites known as essential oils (EOs) which have a wide range of biochemical and physiological functions [163]. The major EO…
Line 464: should read…loss reduction and shelf-life extension…
Line 470-471: should read…efficacy [167].
Line 474-475: should read…even eliminate them.
Line 490-492: Please reword this sentence. It is awkward and difficult to understand the meaning.
Line 496: should read…by-products might also offer a useful solution to the fungus problem…
Line 503-507: should read… Guan et al. [187] reported that, Stenotrophomona smaltophilia displayed a reducing effect on AFB1 (82.5%) when incubated at 37 0 C for 72 hr. After this treatment, that has been shown to affect enzymatic activity, the reaction efficiency significantly dropped, which indicates that AFB1 probably decreased due to enzymatic degradation. (Please make sure this is correct and maybe add a reference.)
Line 544-546: should read…organism (therefore, tagged as GRAS). The fact that they are easily cultured and maintained is also to their advantage…
Line 546-548: should read…Although research on applicability and efficiency of LABs to reduce mould and mycotoxin contamination in different food matrices is challenging and time consuming, it is worth exploring [100].
Line 549-550: should read…the first makes use of viable microbial cells and the second makes use of enzymes…
Line 554-555: should read…such as a cell…
Line 579: should read…aqueous solutions…
Line 597-598: should read…with PBS buffer, because toxin–microbe complexes release approximately 25% to 40% of…
Line 600-601: should read…fermenting microorganisms, therefore in real-life production, biological adsorption usually occurs during fermentation.
Line 610: should read…In the past several years, more attention has been focused on animal polysaccharides. (For?? The sentence is incomplete.)
Line 618: should read…in the adsorption process. Due to the presumed environmental and health acceptability of…
Line 619-621: should read…have attracted more attention. In addition, the reduction of mycotoxins during the production of fermented products, the use of slightly contaminated raw materials would be acceptable.
Line 627: should read…are low cost, effect on a broad spectrum of target mycotoxins,…
Line 635: should read…perceived harmlessness to humans…
Line 641-645: should read…It should be noted, that most of the studies regarding mycotoxin biocontrol completed to date, were conducted under laboratory or simple system conditions. Therefore, more attention should be dedicated to the complete evaluation of practical uses of bio-controlling organisms or their enzymes in field and industrial settings, and storage environments.
Author Response
Comment: This review paper summarizes existing biocontrol methods aimed at reduction and/or prevention of mycotoxins and their associated fungi found predominantly in/on cereal seeds. Included are the most relevant mycotoxins and their respective toxic effects in specific commodities. Pre-harvest biocontrol methods and post-harvest biocontrol methods are discussed. The authors hope the information may be helpful in increasing sustainability of agriculture pertaining to food and feed production. The information is current and relevant, addressing worldwide mycotoxin concerns and should be of interest to a wide audience.
The manuscript is relatively well written, needing minor stylistic edits and inclusion of few details for clarity. Please see the specific comments section.
Answer: We thank the Reviewer for these very helpful comments. In the revised version of the manuscript we have tried to take into account all the mentioned comments/suggestions and appreciate that the quality of the manuscript has been improved significantly.
Specific comments/suggestions:
Comment: Line 46: should read…are viewed as one of the most dangerous…
Answer: The change has been made as suggested.
Comment: Line 47-48: should read…Mycotoxins are secondary metabolites of mostly fungal genera, including the Aspergillus, Fusarium, and Penicillium species.
Answer: The change has been made as suggested.
Comment: Line 54: should read…fungal infestation…
Answer: The change has been made as suggested.
Comment: Line 57: should read…the accumulation of toxic substances.
Answer: The change has been made as suggested.
Comment: Line 64: should read…easily attack damaged kernels,
Answer: The change was made as suggested.
Comment: Line 68: should read…challenges in successful crop…
Answer: The change has been made as suggested.
Comment: Line 69: should read…seedling blight…
Answer: The change was made as suggested.
Comment: Line 72: should read…disease management.
Answer: The change has been made as suggested.
Comment: Line 81-82: should read…can play a key role in minimizing mycotoxin contamination by controlling mould growth and subsequent mycotoxin biosynthesis.
Answer: The change was made as suggested.
Comment: Line 92-96: should read…Moreover, the economic aspects of measures taken against moulds and mycotoxins are not negligible and cannot be overlooked. As a result of climatic changes, these measures must be adapted in order to remain efficient, but need to also be cost effective [21-23]. Given that biocontrol methods use natural sources and do not require additional care after being used, they are considered promising. (From what perspective are they considered promising? Economic perspective?? They may be costly to develop. This may need clarification.)
Answer: The sentence has been expanded in line with the reviewer's comments and we hope it is now written more fully and clearly.
Comment: Line 97: should read…possibility of implementing biocontrol…
Answer: The change has been made as suggested.
Comment: Line 99-100: should read…with mycotoxins, to the ultimate goal of 99 increasing the sustainability of the agricultural chain in food and feed production.
Answer: The change has been made as suggested.
Comment: Line 103: should read…in an accumulative process. Their…
Answer: The change has been made as suggested.
Comment: Line 116: should read…has been modified
Answer: The change has been made as suggested.
Comment: Line 120: should read…Pre-harvest biocontrol methods
Answer: The word „biocontrol“ has been added.
Comment: Line 123-126: should read…Innovations introduced in the pre-harvest stage can be attributed to plant genetic engineering as a biotechnology advancement in mould-produced mycotoxin control. Plant genetic engineering aimed at the development of mycotoxin-resistant varieties and crop detoxification, places the emphasis on the fungi and mycotoxins most relevant from…
Answer: The change has been made as suggested.
Comment: Line 128-129: should read…The most reasonable approach to the prevention of mycotoxin contamination is by breeding cereals and other crops resistant to moulds and therefore not prone to accumulating mycotoxins.
Answer: The change has been made as suggested.
Comment: Line 136: should read…to infection by mycotoxin producing fungi. (Mycotoxins do not infect, they accumulate…fungi infect or colonize and produce mycotoxins. Did you mean ‘mycotoxin accumulation’? This is an important distinction. Need to clarify!)
Answer: We thank the Reviewer for improving the terminology. The change has been made as suggested.
Comment: Line 147-148: should read…a milder infection, and lower mycotoxin levels, in the harvested product…
Answer: This sentence has been removed because almost the entire paragraph has been deleted and replaced with a Table.
Comment: Line 149: should read…in maize ears;
Comment: Line 151-152: should read…to create a transgenic maize line. Upon subsequent inoculation with Aspergillus flavus sufficiently concentrated to infect the transgenic maize ears, AF was not detected…(please use ‘ears’ instead of ‘cobs’)
Line 154: …gave high protection levels in field trials…(against what?? Fusarium? Fumonisin?? Please complete the sentence)
Line 156: …reduced disease severity (what disease? Please clarify)
Line 159-160: …this disease… (what disease? Please clarify)
Line 161-162: incomplete sentence…should perhaps read??... Karlovsky [55], Duvick [56], and Munkvold [57] reviewed a variety of genetic-based approaches that are being or have been used for mycotoxin control. (Please make sure the sentence reads as you intended, if not, please correct it.)
Line 162: should read… One of the approaches…
Line 163: reduce infection…(fungal infection? Please clarify.)
Line 179: should read… as compared to the parent maize.
Line 182: Additionally, reductions of aflatoxin production in Bt hybrids were not observed. (Are you sure? Do you have a reference for this statement??)
Answer: These sentences have been removed in the revised version of the manuscript because almost the entire paragraph has been deleted and replaced with a Table (also in accordance with the opinion of another Reviewer).
Comment: Line 186: should read… remains controversial.
Answer: The change has been made as suggested.
Comment: Lines 205-206: should read… In the last few decades, the interest in PBM use for increasing yields and resilience of agricultural crops has been steadily growing…
Answer: The change has been made as suggested.
Comment: Lines 213-214: should read… Increase in grain yield and stability was associated with seed coating formulation with the AM fungi and Trichoderma consortium with reduced or withheld fertilizer…
Answer: The change has been made as suggested.
Comment: Line 223: should read…to maintain crop health and vigor.
Answer: The change has been made as suggested.
Comment: Line 328: should read… Abbas…
Answer: The change has been made as suggested.
Comment: Line 345: should read… maize was also reported [131]. Additionally, the inhibitory effect…
Answer: The change has been made as suggested.
Comment: Line 368: should read… nontoxigenic F. verticillioides…
Answer: The change has been made as suggested.
Comment: Line 369-370: should read… and inhibiting toxin synthesis.
Answer: The change has been made as suggested.
Comment: Line 373-374: should read… which resulted in prevention of accumulation of mycotoxins during storage.
Answer: The change has been made as suggested.
Comment: Line 384: should read… Post-harvest biocontrol methods
Answer: The change has been made as suggested.
Comment: Line 388-390: should read… is that they do not significantly change the physicochemical properties of the cereals and leave any toxic mycotoxin residue behind [121,147]. In addition, the cost of …
Answer: The change has been made as suggested.
Comment: Line 392: should read… more mycotoxin…
Answer: The change has been made as suggested.
Comment: Line 396: should read… if that is not possible, one has to ensure to…
Answer: The change has been made as suggested.
Comment: Line 418: should read…affected the conidia producing ability of the fungus.
Answer: The change has been made as suggested.
Comment: Line 422: should read…inhibited the growth…
Answer: The change has been made as suggested.
Comment: Line 423-424: should read…Evidence from microscopy and quantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that,…
Answer: The change has been made as suggested.
Comment: Line 433: What are ‘cellular laches’?
Answer: The term which authors of cited reference used has now been removed, since we are not sure that the mentioned term would be clearly understood.
Comment: Line 439: should read…phenolic compounds,…
Answer: The change has been made as suggested.
Comment: Line 444: should read…type of acid, concentration, the…
Answer: The change has been made as suggested.
Comment: Line 452: should read…reported antifungal…
Answer: The change has been made as suggested.
Comment: Line 455: should read…needed to elucidate the mechanism…
Answer: The change has been made as suggested.
Comment: Line 458-459: should read…Some higher plants produce secondary metabolites known as essential oils (EOs) which have a wide range of biochemical and physiological functions [163]. The major EO…
Answer: The change has been made as suggested.
Comment: Line 464: should read…loss reduction and shelf-life extension…
Answer: The change has been made as suggested.
Comment: Line 470-471: should read…efficacy [167].
Answer: The change has been made as suggested.
Comment: Line 474-475: should read…even eliminate them.
Answer: The change has been made as suggested.
Comment: Line 490-492: Please reword this sentence. It is awkward and difficult to understand the meaning.
Answer: The sentence has been reformulated.
Comment: Line 496: should read…by-products might also offer a useful solution to the fungus problem…
Answer: The change has been made as suggested.
Comment: Line 503-507: should read… Guan et al. [187] reported that, Stenotrophomona smaltophilia displayed a reducing effect on AFB1 (82.5%) when incubated at 37 0 C for 72 hr. After this treatment, that has been shown to affect enzymatic activity, the reaction efficiency significantly dropped, which indicates that AFB1 probably decreased due to enzymatic degradation. (Please make sure this is correct and maybe add a reference.)
Answer: It has been corrected. A reference is not necessary because this claim came from the research of Guan et al.
Comment: Line 544-546: should read…organism (therefore, tagged as GRAS). The fact that they are easily cultured and maintained is also to their advantage…
Answer: The change has been made as suggested.
Comment: Line 546-548: should read…Although research on applicability and efficiency of LABs to reduce mould and mycotoxin contamination in different food matrices is challenging and time consuming, it is worth exploring [100].
Answer: The change has been made as suggested.
Comment: Line 549-550: should read…the first makes use of viable microbial cells and the second makes use of enzymes…
Answer: The change has been made as suggested.
Comment: Line 554-555: should read…such as a cell…
Answer: The change has been made as suggested.
Comment: Line 579: should read…aqueous solutions…
Answer: The word watery has been changed as suggested.
Comment: Line 597-598: should read…with PBS buffer, because toxin–microbe complexes release approximately 25% to 40% of…
Answer: The change has been made as suggested.
Comment: Line 600-601: should read…fermenting microorganisms, therefore in real-life production, biological adsorption usually occurs during fermentation.
Answer: The change has been made as suggested.
Comment: Line 610: should read…In the past several years, more attention has been focused on animal polysaccharides. (For?? The sentence is incomplete.)
Answer: The sentence has been corrected and completed with an explanation of how animal polysaccharides can be used in mycotoxins adsorption.
Comment: Line 618: should read…in the adsorption process. Due to the presumed environmental and health acceptability of…
Answer: The change has been made as suggested.
Comment: Line 619-621: should read…have attracted more attention. In addition, the reduction of mycotoxins during the production of fermented products, the use of slightly contaminated raw materials would be acceptable.
Answer: The sentence has been written as the Reviewer suggested.
Comment: Line 627: should read…are low cost, effect on a broad spectrum of target mycotoxins,…
Answer: The change has been made as suggested.
Comment: Line 635: should read…perceived harmlessness to humans…
Answer: The word perceived has been added.
Comment: Line 641-645: should read…It should be noted, that most of the studies regarding mycotoxin biocontrol completed to date, were conducted under laboratory or simple system conditions. Therefore, more attention should be dedicated to the complete evaluation of practical uses of bio-controlling organisms or their enzymes in field and industrial settings, and storage environments.
Answer: We thank the Reviewer for all their useful remarks. The sentence is now written as the Reviewer suggested.
The authors are indebted to our esteemed Reviewer for his/her most helpful suggestions and comments. In the revised version, changes to the original manuscript are marked in track changes mode.
Comments to the Referee 1:
Comment: This review paper summarizes existing biocontrol methods aimed at reduction and/or prevention of mycotoxins and their associated fungi found predominantly in/on cereal seeds. Included are the most relevant mycotoxins and their respective toxic effects in specific commodities. Pre-harvest biocontrol methods and post-harvest biocontrol methods are discussed. The authors hope the information may be helpful in increasing sustainability of agriculture pertaining to food and feed production. The information is current and relevant, addressing worldwide mycotoxin concerns and should be of interest to a wide audience.
The manuscript is relatively well written, needing minor stylistic edits and inclusion of few details for clarity. Please see the specific comments section.
Answer: We thank the Reviewer for these very helpful comments. In the revised version of the manuscript we have tried to take into account all the mentioned comments/suggestions and appreciate that the quality of the manuscript has been improved significantly.
Specific comments/suggestions:
Comment: Line 46: should read…are viewed as one of the most dangerous…
Answer: The change has been made as suggested.
Comment: Line 47-48: should read…Mycotoxins are secondary metabolites of mostly fungal genera, including the Aspergillus, Fusarium, and Penicillium species.
Answer: The change has been made as suggested.
Comment: Line 54: should read…fungal infestation…
Answer: The change has been made as suggested.
Comment: Line 57: should read…the accumulation of toxic substances.
Answer: The change has been made as suggested.
Comment: Line 64: should read…easily attack damaged kernels,
Answer: The change was made as suggested.
Comment: Line 68: should read…challenges in successful crop…
Answer: The change has been made as suggested.
Comment: Line 69: should read…seedling blight…
Answer: The change was made as suggested.
Comment: Line 72: should read…disease management.
Answer: The change has been made as suggested.
Comment: Line 81-82: should read…can play a key role in minimizing mycotoxin contamination by controlling mould growth and subsequent mycotoxin biosynthesis.
Answer: The change was made as suggested.
Comment: Line 92-96: should read…Moreover, the economic aspects of measures taken against moulds and mycotoxins are not negligible and cannot be overlooked. As a result of climatic changes, these measures must be adapted in order to remain efficient, but need to also be cost effective [21-23]. Given that biocontrol methods use natural sources and do not require additional care after being used, they are considered promising. (From what perspective are they considered promising? Economic perspective?? They may be costly to develop. This may need clarification.)
Answer: The sentence has been expanded in line with the reviewer's comments and we hope it is now written more fully and clearly.
Comment: Line 97: should read…possibility of implementing biocontrol…
Answer: The change has been made as suggested.
Comment: Line 99-100: should read…with mycotoxins, to the ultimate goal of 99 increasing the sustainability of the agricultural chain in food and feed production.
Answer: The change has been made as suggested.
Comment: Line 103: should read…in an accumulative process. Their…
Answer: The change has been made as suggested.
Comment: Line 116: should read…has been modified
Answer: The change has been made as suggested.
Comment: Line 120: should read…Pre-harvest biocontrol methods
Answer: The word „biocontrol“ has been added.
Comment: Line 123-126: should read…Innovations introduced in the pre-harvest stage can be attributed to plant genetic engineering as a biotechnology advancement in mould-produced mycotoxin control. Plant genetic engineering aimed at the development of mycotoxin-resistant varieties and crop detoxification, places the emphasis on the fungi and mycotoxins most relevant from…
Answer: The change has been made as suggested.
Comment: Line 128-129: should read…The most reasonable approach to the prevention of mycotoxin contamination is by breeding cereals and other crops resistant to moulds and therefore not prone to accumulating mycotoxins.
Answer: The change has been made as suggested.
Comment: Line 136: should read…to infection by mycotoxin producing fungi. (Mycotoxins do not infect, they accumulate…fungi infect or colonize and produce mycotoxins. Did you mean ‘mycotoxin accumulation’? This is an important distinction. Need to clarify!)
Answer: We thank the Reviewer for improving the terminology. The change has been made as suggested.
Comment: Line 147-148: should read…a milder infection, and lower mycotoxin levels, in the harvested product…
Answer: This sentence has been removed because almost the entire paragraph has been deleted and replaced with a Table.
Comment: Line 149: should read…in maize ears;
Comment: Line 151-152: should read…to create a transgenic maize line. Upon subsequent inoculation with Aspergillus flavus sufficiently concentrated to infect the transgenic maize ears, AF was not detected…(please use ‘ears’ instead of ‘cobs’)
Line 154: …gave high protection levels in field trials…(against what?? Fusarium? Fumonisin?? Please complete the sentence)
Line 156: …reduced disease severity (what disease? Please clarify)
Line 159-160: …this disease… (what disease? Please clarify)
Line 161-162: incomplete sentence…should perhaps read??... Karlovsky [55], Duvick [56], and Munkvold [57] reviewed a variety of genetic-based approaches that are being or have been used for mycotoxin control. (Please make sure the sentence reads as you intended, if not, please correct it.)
Line 162: should read… One of the approaches…
Line 163: reduce infection…(fungal infection? Please clarify.)
Line 179: should read… as compared to the parent maize.
Line 182: Additionally, reductions of aflatoxin production in Bt hybrids were not observed. (Are you sure? Do you have a reference for this statement??)
Answer: These sentences have been removed in the revised version of the manuscript because almost the entire paragraph has been deleted and replaced with a Table (also in accordance with the opinion of another Reviewer).
Comment: Line 186: should read… remains controversial.
Answer: The change has been made as suggested.
Comment: Lines 205-206: should read… In the last few decades, the interest in PBM use for increasing yields and resilience of agricultural crops has been steadily growing…
Answer: The change has been made as suggested.
Comment: Lines 213-214: should read… Increase in grain yield and stability was associated with seed coating formulation with the AM fungi and Trichoderma consortium with reduced or withheld fertilizer…
Answer: The change has been made as suggested.
Comment: Line 223: should read…to maintain crop health and vigor.
Answer: The change has been made as suggested.
Comment: Line 328: should read… Abbas…
Answer: The change has been made as suggested.
Comment: Line 345: should read… maize was also reported [131]. Additionally, the inhibitory effect…
Answer: The change has been made as suggested.
Comment: Line 368: should read… nontoxigenic F. verticillioides…
Answer: The change has been made as suggested.
Comment: Line 369-370: should read… and inhibiting toxin synthesis.
Answer: The change has been made as suggested.
Comment: Line 373-374: should read… which resulted in prevention of accumulation of mycotoxins during storage.
Answer: The change has been made as suggested.
Comment: Line 384: should read… Post-harvest biocontrol methods
Answer: The change has been made as suggested.
Comment: Line 388-390: should read… is that they do not significantly change the physicochemical properties of the cereals and leave any toxic mycotoxin residue behind [121,147]. In addition, the cost of …
Answer: The change has been made as suggested.
Comment: Line 392: should read… more mycotoxin…
Answer: The change has been made as suggested.
Comment: Line 396: should read… if that is not possible, one has to ensure to…
Answer: The change has been made as suggested.
Comment: Line 418: should read…affected the conidia producing ability of the fungus.
Answer: The change has been made as suggested.
Comment: Line 422: should read…inhibited the growth…
Answer: The change has been made as suggested.
Comment: Line 423-424: should read…Evidence from microscopy and quantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that,…
Answer: The change has been made as suggested.
Comment: Line 433: What are ‘cellular laches’?
Answer: The term which authors of cited reference used has now been removed, since we are not sure that the mentioned term would be clearly understood.
Comment: Line 439: should read…phenolic compounds,…
Answer: The change has been made as suggested.
Comment: Line 444: should read…type of acid, concentration, the…
Answer: The change has been made as suggested.
Comment: Line 452: should read…reported antifungal…
Answer: The change has been made as suggested.
Comment: Line 455: should read…needed to elucidate the mechanism…
Answer: The change has been made as suggested.
Comment: Line 458-459: should read…Some higher plants produce secondary metabolites known as essential oils (EOs) which have a wide range of biochemical and physiological functions [163]. The major EO…
Answer: The change has been made as suggested.
Comment: Line 464: should read…loss reduction and shelf-life extension…
Answer: The change has been made as suggested.
Comment: Line 470-471: should read…efficacy [167].
Answer: The change has been made as suggested.
Comment: Line 474-475: should read…even eliminate them.
Answer: The change has been made as suggested.
Comment: Line 490-492: Please reword this sentence. It is awkward and difficult to understand the meaning.
Answer: The sentence has been reformulated.
Comment: Line 496: should read…by-products might also offer a useful solution to the fungus problem…
Answer: The change has been made as suggested.
Comment: Line 503-507: should read… Guan et al. [187] reported that, Stenotrophomona smaltophilia displayed a reducing effect on AFB1 (82.5%) when incubated at 37 0 C for 72 hr. After this treatment, that has been shown to affect enzymatic activity, the reaction efficiency significantly dropped, which indicates that AFB1 probably decreased due to enzymatic degradation. (Please make sure this is correct and maybe add a reference.)
Answer: It has been corrected. A reference is not necessary because this claim came from the research of Guan et al.
Comment: Line 544-546: should read…organism (therefore, tagged as GRAS). The fact that they are easily cultured and maintained is also to their advantage…
Answer: The change has been made as suggested.
Comment: Line 546-548: should read…Although research on applicability and efficiency of LABs to reduce mould and mycotoxin contamination in different food matrices is challenging and time consuming, it is worth exploring [100].
Answer: The change has been made as suggested.
Comment: Line 549-550: should read…the first makes use of viable microbial cells and the second makes use of enzymes…
Answer: The change has been made as suggested.
Comment: Line 554-555: should read…such as a cell…
Answer: The change has been made as suggested.
Comment: Line 579: should read…aqueous solutions…
Answer: The word watery has been changed as suggested.
Comment: Line 597-598: should read…with PBS buffer, because toxin–microbe complexes release approximately 25% to 40% of…
Answer: The change has been made as suggested.
Comment: Line 600-601: should read…fermenting microorganisms, therefore in real-life production, biological adsorption usually occurs during fermentation.
Answer: The change has been made as suggested.
Comment: Line 610: should read…In the past several years, more attention has been focused on animal polysaccharides. (For?? The sentence is incomplete.)
Answer: The sentence has been corrected and completed with an explanation of how animal polysaccharides can be used in mycotoxins adsorption.
Comment: Line 618: should read…in the adsorption process. Due to the presumed environmental and health acceptability of…
Answer: The change has been made as suggested.
Comment: Line 619-621: should read…have attracted more attention. In addition, the reduction of mycotoxins during the production of fermented products, the use of slightly contaminated raw materials would be acceptable.
Answer: The sentence has been written as the Reviewer suggested.
Comment: Line 627: should read…are low cost, effect on a broad spectrum of target mycotoxins,…
Answer: The change has been made as suggested.
Comment: Line 635: should read…perceived harmlessness to humans…
Answer: The word perceived has been added.
Comment: Line 641-645: should read…It should be noted, that most of the studies regarding mycotoxin biocontrol completed to date, were conducted under laboratory or simple system conditions. Therefore, more attention should be dedicated to the complete evaluation of practical uses of bio-controlling organisms or their enzymes in field and industrial settings, and storage environments.
Answer: We thank the Reviewer for all their useful remarks. The sentence is now written as the Reviewer suggested.
The authors are indebted to our esteemed Reviewer for his/her most helpful suggestions and comments. In the revised version, changes to the original manuscript are marked in track changes mode.
Comments to the Referee 1:
Comment: This review paper summarizes existing biocontrol methods aimed at reduction and/or prevention of mycotoxins and their associated fungi found predominantly in/on cereal seeds. Included are the most relevant mycotoxins and their respective toxic effects in specific commodities. Pre-harvest biocontrol methods and post-harvest biocontrol methods are discussed. The authors hope the information may be helpful in increasing sustainability of agriculture pertaining to food and feed production. The information is current and relevant, addressing worldwide mycotoxin concerns and should be of interest to a wide audience.
The manuscript is relatively well written, needing minor stylistic edits and inclusion of few details for clarity. Please see the specific comments section.
Answer: We thank the Reviewer for these very helpful comments. In the revised version of the manuscript we have tried to take into account all the mentioned comments/suggestions and appreciate that the quality of the manuscript has been improved significantly.
Specific comments/suggestions:
Comment: Line 46: should read…are viewed as one of the most dangerous…
Answer: The change has been made as suggested.
Comment: Line 47-48: should read…Mycotoxins are secondary metabolites of mostly fungal genera, including the Aspergillus, Fusarium, and Penicillium species.
Answer: The change has been made as suggested.
Comment: Line 54: should read…fungal infestation…
Answer: The change has been made as suggested.
Comment: Line 57: should read…the accumulation of toxic substances.
Answer: The change has been made as suggested.
Comment: Line 64: should read…easily attack damaged kernels,
Answer: The change was made as suggested.
Comment: Line 68: should read…challenges in successful crop…
Answer: The change has been made as suggested.
Comment: Line 69: should read…seedling blight…
Answer: The change was made as suggested.
Comment: Line 72: should read…disease management.
Answer: The change has been made as suggested.
Comment: Line 81-82: should read…can play a key role in minimizing mycotoxin contamination by controlling mould growth and subsequent mycotoxin biosynthesis.
Answer: The change was made as suggested.
Comment: Line 92-96: should read…Moreover, the economic aspects of measures taken against moulds and mycotoxins are not negligible and cannot be overlooked. As a result of climatic changes, these measures must be adapted in order to remain efficient, but need to also be cost effective [21-23]. Given that biocontrol methods use natural sources and do not require additional care after being used, they are considered promising. (From what perspective are they considered promising? Economic perspective?? They may be costly to develop. This may need clarification.)
Answer: The sentence has been expanded in line with the reviewer's comments and we hope it is now written more fully and clearly.
Comment: Line 97: should read…possibility of implementing biocontrol…
Answer: The change has been made as suggested.
Comment: Line 99-100: should read…with mycotoxins, to the ultimate goal of 99 increasing the sustainability of the agricultural chain in food and feed production.
Answer: The change has been made as suggested.
Comment: Line 103: should read…in an accumulative process. Their…
Answer: The change has been made as suggested.
Comment: Line 116: should read…has been modified
Answer: The change has been made as suggested.
Comment: Line 120: should read…Pre-harvest biocontrol methods
Answer: The word „biocontrol“ has been added.
Comment: Line 123-126: should read…Innovations introduced in the pre-harvest stage can be attributed to plant genetic engineering as a biotechnology advancement in mould-produced mycotoxin control. Plant genetic engineering aimed at the development of mycotoxin-resistant varieties and crop detoxification, places the emphasis on the fungi and mycotoxins most relevant from…
Answer: The change has been made as suggested.
Comment: Line 128-129: should read…The most reasonable approach to the prevention of mycotoxin contamination is by breeding cereals and other crops resistant to moulds and therefore not prone to accumulating mycotoxins.
Answer: The change has been made as suggested.
Comment: Line 136: should read…to infection by mycotoxin producing fungi. (Mycotoxins do not infect, they accumulate…fungi infect or colonize and produce mycotoxins. Did you mean ‘mycotoxin accumulation’? This is an important distinction. Need to clarify!)
Answer: We thank the Reviewer for improving the terminology. The change has been made as suggested.
Comment: Line 147-148: should read…a milder infection, and lower mycotoxin levels, in the harvested product…
Answer: This sentence has been removed because almost the entire paragraph has been deleted and replaced with a Table.
Comment: Line 149: should read…in maize ears;
Comment: Line 151-152: should read…to create a transgenic maize line. Upon subsequent inoculation with Aspergillus flavus sufficiently concentrated to infect the transgenic maize ears, AF was not detected…(please use ‘ears’ instead of ‘cobs’)
Line 154: …gave high protection levels in field trials…(against what?? Fusarium? Fumonisin?? Please complete the sentence)
Line 156: …reduced disease severity (what disease? Please clarify)
Line 159-160: …this disease… (what disease? Please clarify)
Line 161-162: incomplete sentence…should perhaps read??... Karlovsky [55], Duvick [56], and Munkvold [57] reviewed a variety of genetic-based approaches that are being or have been used for mycotoxin control. (Please make sure the sentence reads as you intended, if not, please correct it.)
Line 162: should read… One of the approaches…
Line 163: reduce infection…(fungal infection? Please clarify.)
Line 179: should read… as compared to the parent maize.
Line 182: Additionally, reductions of aflatoxin production in Bt hybrids were not observed. (Are you sure? Do you have a reference for this statement??)
Answer: These sentences have been removed in the revised version of the manuscript because almost the entire paragraph has been deleted and replaced with a Table (also in accordance with the opinion of another Reviewer).
Comment: Line 186: should read… remains controversial.
Answer: The change has been made as suggested.
Comment: Lines 205-206: should read… In the last few decades, the interest in PBM use for increasing yields and resilience of agricultural crops has been steadily growing…
Answer: The change has been made as suggested.
Comment: Lines 213-214: should read… Increase in grain yield and stability was associated with seed coating formulation with the AM fungi and Trichoderma consortium with reduced or withheld fertilizer…
Answer: The change has been made as suggested.
Comment: Line 223: should read…to maintain crop health and vigor.
Answer: The change has been made as suggested.
Comment: Line 328: should read… Abbas…
Answer: The change has been made as suggested.
Comment: Line 345: should read… maize was also reported [131]. Additionally, the inhibitory effect…
Answer: The change has been made as suggested.
Comment: Line 368: should read… nontoxigenic F. verticillioides…
Answer: The change has been made as suggested.
Comment: Line 369-370: should read… and inhibiting toxin synthesis.
Answer: The change has been made as suggested.
Comment: Line 373-374: should read… which resulted in prevention of accumulation of mycotoxins during storage.
Answer: The change has been made as suggested.
Comment: Line 384: should read… Post-harvest biocontrol methods
Answer: The change has been made as suggested.
Comment: Line 388-390: should read… is that they do not significantly change the physicochemical properties of the cereals and leave any toxic mycotoxin residue behind [121,147]. In addition, the cost of …
Answer: The change has been made as suggested.
Comment: Line 392: should read… more mycotoxin…
Answer: The change has been made as suggested.
Comment: Line 396: should read… if that is not possible, one has to ensure to…
Answer: The change has been made as suggested.
Comment: Line 418: should read…affected the conidia producing ability of the fungus.
Answer: The change has been made as suggested.
Comment: Line 422: should read…inhibited the growth…
Answer: The change has been made as suggested.
Comment: Line 423-424: should read…Evidence from microscopy and quantitative reverse transcription polymerase chain reaction (RT-PCR) indicate that,…
Answer: The change has been made as suggested.
Comment: Line 433: What are ‘cellular laches’?
Answer: The term which authors of cited reference used has now been removed, since we are not sure that the mentioned term would be clearly understood.
Comment: Line 439: should read…phenolic compounds,…
Answer: The change has been made as suggested.
Comment: Line 444: should read…type of acid, concentration, the…
Answer: The change has been made as suggested.
Comment: Line 452: should read…reported antifungal…
Answer: The change has been made as suggested.
Comment: Line 455: should read…needed to elucidate the mechanism…
Answer: The change has been made as suggested.
Comment: Line 458-459: should read…Some higher plants produce secondary metabolites known as essential oils (EOs) which have a wide range of biochemical and physiological functions [163]. The major EO…
Answer: The change has been made as suggested.
Comment: Line 464: should read…loss reduction and shelf-life extension…
Answer: The change has been made as suggested.
Comment: Line 470-471: should read…efficacy [167].
Answer: The change has been made as suggested.
Comment: Line 474-475: should read…even eliminate them.
Answer: The change has been made as suggested.
Comment: Line 490-492: Please reword this sentence. It is awkward and difficult to understand the meaning.
Answer: The sentence has been reformulated.
Comment: Line 496: should read…by-products might also offer a useful solution to the fungus problem…
Answer: The change has been made as suggested.
Comment: Line 503-507: should read… Guan et al. [187] reported that, Stenotrophomona smaltophilia displayed a reducing effect on AFB1 (82.5%) when incubated at 37 0 C for 72 hr. After this treatment, that has been shown to affect enzymatic activity, the reaction efficiency significantly dropped, which indicates that AFB1 probably decreased due to enzymatic degradation. (Please make sure this is correct and maybe add a reference.)
Answer: It has been corrected. A reference is not necessary because this claim came from the research of Guan et al.
Comment: Line 544-546: should read…organism (therefore, tagged as GRAS). The fact that they are easily cultured and maintained is also to their advantage…
Answer: The change has been made as suggested.
Comment: Line 546-548: should read…Although research on applicability and efficiency of LABs to reduce mould and mycotoxin contamination in different food matrices is challenging and time consuming, it is worth exploring [100].
Answer: The change has been made as suggested.
Comment: Line 549-550: should read…the first makes use of viable microbial cells and the second makes use of enzymes…
Answer: The change has been made as suggested.
Comment: Line 554-555: should read…such as a cell…
Answer: The change has been made as suggested.
Comment: Line 579: should read…aqueous solutions…
Answer: The word watery has been changed as suggested.
Comment: Line 597-598: should read…with PBS buffer, because toxin–microbe complexes release approximately 25% to 40% of…
Answer: The change has been made as suggested.
Comment: Line 600-601: should read…fermenting microorganisms, therefore in real-life production, biological adsorption usually occurs during fermentation.
Answer: The change has been made as suggested.
Comment: Line 610: should read…In the past several years, more attention has been focused on animal polysaccharides. (For?? The sentence is incomplete.)
Answer: The sentence has been corrected and completed with an explanation of how animal polysaccharides can be used in mycotoxins adsorption.
Comment: Line 618: should read…in the adsorption process. Due to the presumed environmental and health acceptability of…
Answer: The change has been made as suggested.
Comment: Line 619-621: should read…have attracted more attention. In addition, the reduction of mycotoxins during the production of fermented products, the use of slightly contaminated raw materials would be acceptable.
Answer: The sentence has been written as the Reviewer suggested.
Comment: Line 627: should read…are low cost, effect on a broad spectrum of target mycotoxins,…
Answer: The change has been made as suggested.
Comment: Line 635: should read…perceived harmlessness to humans…
Answer: The word perceived has been added.
Comment: Line 641-645: should read…It should be noted, that most of the studies regarding mycotoxin biocontrol completed to date, were conducted under laboratory or simple system conditions. Therefore, more attention should be dedicated to the complete evaluation of practical uses of bio-controlling organisms or their enzymes in field and industrial settings, and storage environments.
Answer: We thank the Reviewer for all their useful remarks. The sentence is now written as the Reviewer suggested.
The authors are indebted to our esteemed Reviewer for his/her most helpful suggestions and comments. In the revised version, changes to the original manuscript are marked in track changes mode.
Reviewer 2 Report
The manuscript “Bio-control methods in prevention and reduction of mycotoxin contamination of seeds and cereals” aims to review biocontrol methods and method prevention and reduction of seeds/cereal mycotoxin contamination.
In my opinion this manuscript looks more like a bibliography collection than a real review. In effect, the manuscript tries to cover too many things at one time, some of these very old, and there is not enough space to deal with problems and solutions in detail. I suggest reorganizing the paper.
As you can find in the instructions of the authors, Reviews “provide concise and precise updates on the latest progress made in a given area of research”. I suggest to focus preferably the paper on the studies of the last 5-10 years.
Additional points of criticism:
- The title is misleading: is the authors’ intention to focus the review not only on cereals, but on every kind of seeds? In this hypothesis (all kind of seeds), the field to explore is too big. On the other hand, the great majority of the examples reported in the text is on cereals.
- The section 3.1.1 reports a mix of studies focused on the identification of resistance genes and plant breeding approaches. The introduction of tables can be of help for a better understanding.
- The review in general can be improved by introducing figures, schemes and/or tables to help the reader.
- A perspective section could be added to individuate the best paths for mycotoxin control.
Other minor comments/corrections:
Line 39: biotic stresses? Drought and salinity are abiotic stresses. Please, correct
Lines 60-61: In my opinion, in the list two very important field fungi are omitted: F.graminearum and F.culmorum. I suggest adding these two species.
Line 109: please, add dot after [27]
Line 112: In my opinion It is not “mode of action” but the “toxic effects”.
Table 1: Please, in “Commodity” correct “Barely, Barly”
Line 117: Figure 1 about “virtue of good agricultural practices” is not present in the paper. Please, add.
Line 119: the reference to Figure 2 about “Pre-harvest and post-harvest methods of bio-control of mycotoxins found in cereals” is not present in the text. Please, add.
Line 217: The authors of reference [78] are Perelló and Dal Bello. Please, correct
Line 328: please, write “abbas” with capital letter
Line 352: what kind of bacterium?
Line 459: please, write “Eo” with capital letters
Lines 468-470 and 471-473: Please, write common name and Latin name in the same order (e.g. Latin name (common name) or common name (Latin name))
Line 488: please, add dot after [178]
Line 504: Please, correct bacterium name “Stenotrophomonas maltophilia”
Lines 505 and 514 and 515: hr or h? Please, write in the same and correct way
Line 609: please, add dot after “Liu et al”
In References: Different errors are present in references. Please, write the references as the journal instructions: “Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range”.
For example:
- in reference 11: eliminate the year after the authors list, write the Abbreviated Journal Name, eliminate comma after the journal name, add space between Volume, page range
- in reference 106: write the Abbreviated Journal Name
- in references 115 and 216: eliminate the year after the authors list
- in reference 221: write the year in bold type
- in reference 224: eliminate the year after the authors list, write the correctly Abbreviated Journal Name
…….
Author Response
Comment: The manuscript “Bio-control methods in prevention and reduction of mycotoxin contamination of seeds and cereals” aims to review biocontrol methods and method prevention and reduction of seeds/cereal mycotoxin contamination. In my opinion this manuscript looks more like a bibliography collection than a real review. In effect, the manuscript tries to cover too many things at one time, some of these very old, and there is not enough space to deal with problems and solutions in detail. I suggest reorganizing the paper. As you can find in the instructions of the authors, Reviews “provide concise and precise updates on the latest progress made in a given area of research”. I suggest to focus preferably the paper on the studies of the last 5-10 years.
Answer: We thank the Reviewer for their very helpful comments. In the revised version of the manuscript we have tried to take into account the above and tried to reorganize it in accordance with the comments of the Reviewer, also using references from the last 5-10 years wherever possible.
Additional points of criticism:
Comment: The title is misleading: is the authors’ intention to focus the review not only on cereals, but on every kind of seeds? In this hypothesis (all kind of seeds), the field to explore is too big. On the other hand, the great majority of the examples reported in the text is on cereals.
Answer: We thank the Reviewer for this very helpful comment. We agree that seed is too broad a field of research and that the content of this paper refers to cereal seeds. The term cereal seeds is now included in the title of the paper.
Comment: The section 3.1.1 reports a mix of studies focused on the identification of resistance genes and plant breeding approaches. The introduction of tables can be of help for a better understanding. The review in general can be improved by introducing figures, schemes and/or tables to help the reader.
Answer: We thank the Reviewer for this very helpful comment. In accordance with the Reviewer's comments, we have created two additional illustrations, that is, one table and one sheme for the sake of clarification of the paper and therefore a better understanding of this issue.
Comment: A perspective section could be added to individuate the best paths for mycotoxin control.
Answer: At the end of each description of biocontrol and contamination, a brief overview of the possibility of the application and effectiveness of the described method is given. If we were to add a separate paragraph to the conclusions, we believe that this would simply be a repetition of what has already been mentioned. Therefore, we have now called the last section of the paper “Conclusions and perspectives” and in this section we have added a few sentences in line with the Reviewer's comment.
Other minor comments/corrections:
Comment: Line 39: biotic stresses? Drought and salinity are abiotic stresses. Please, correct
Answer: The mistake made has been corrected and now these terms are listed as abiotic stresses.
Comment: Lines 60-61: In my opinion, in the list two very important field fungi are omitted: F.graminearum and F. culmorum. I suggest adding these two species.
Answer: We thank the Reviewer for this very helpful comment. These types of moulds from the genus Fusarium have now been added to the text.
Comment: Line 109: please, add dot after [27]
Answer: The dot has now been added.
Comment: Line 112: In my opinion It is not “mode of action” but the “toxic effects”.
Answer: We thank the Reviewer for this useful remark and the term "toxic effects" has now been replaced by the term "mode of action".
Comment: Table 1: Please, in “Commodity” correct “Barely, Barly”
Answer: The misspelled word is now corrected.
Comment: Line 117: Figure 1 about “virtue of good agricultural practices” is not present in the paper. Please, add.
Answer: The error has been corrected and Figure 1 is now mentioned in the paper.
Comment: Line 119: the reference to Figure 2 about “Pre-harvest and post-harvest methods of bio-control of mycotoxins found in cereals” is not present in the text. Please, add.
Answer: We inadvertently marked Figure 1 as Figure 2. Figure 2 was not present in the paper before, and now the citation of images in the paper has been corrected, at the same time with the inclusion of newly added figures.
Comment: Line 217: The authors of reference [78] are Perelló and Dal Bello. Please, correct.
Answer: The listed authors’ names have now been corrected.
Comment: Line 328: please, write “abbas” with capital letter
Answer: Abbas is now written with a capital letter.
Comment: Line 352: what kind of bacterium?
Answer: Since several types of bacteria are discussed after, a "different kind of bacterium species" has now been added to the text.
Comment: Line 459: please, write “Eo” with capital letters
Answer: EO is now written in capital letters.
Comment: Lines 468-470 and 471-473: Please, write common name and Latin name in the same order (e.g. Latin name (common name) or common name (Latin name))
Line 488: please, add dot after [178]
Answer: The dot has now been added.
Comment: Line 504: Please, correct bacterium name “Stenotrophomonas maltophilia”
Answer: The name of the bacteria is corrected.
Comment: Lines 505 and 514 and 515: hr or h? Please, write in the same and correct way.
Answer: Abbreviation “h” for hours is now written uniformly.
Comment: Line 609: please, add dot after “Liu et al”
Answer: The dot has now been added.
Comment: In References: Different errors are present in references. Please, write the references as the journal instructions: “Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range”.
For example:
- in reference 11: eliminate the year after the authors list, write the Abbreviated Journal Name, eliminate comma after the journal name, add space between Volume, page range
Answer: The necessary changes have been made.
- in reference 106: write the Abbreviated Journal Name
Answer: Abbreviated journal name is now written.
- in references 115 and 216: eliminate the year after the authors list
Answer: The year has now been deleted.
- in reference 221: write the year in bold type
Answer: The reference has been corrected.
- in reference 224: eliminate the year after the authors list, write the correctly Abbreviated Journal Name
Answer: The year is now eliminated and the abbreviation for journal is now written.
The authors are indebted to our esteemed Reviewer for his/her most helpful suggestions and comments. In the revised version, changes to the original manuscript are marked in track changes mode.
Round 2
Reviewer 2 Report
Thanks for improving the manuscript, now it is ready for publication